Poly-.beta.-hydroxybutyrate "PHB" is a naturally occurring bacterial polyester that was discovered by Lemoigne in 1926 (Lemoigne, Bull. Soc. Chim. Biol. 8:770, 1926). PHB is believed to exist as a bacterial energy storage compound which is accumulated during times of nutritional stress, and is degraded when the stress is relieved (Oeding et al., Biochemical Journal 134:239-248, 1973; Senior et al., Biochemistry Journal 134:225-238, 1973). The most remarkable aspect of PHB accumulation is the intracellular levels to which it can accumulate. In Alcaligenes eutrophus, PHB levels have been known to reach 80% of the cell dry weight (Oeding et al., Biochemical Journal 134:239-248, 1973).
In the early 1950's it was discovered that purified PHB was, in fact, a biodegradable thermoplastic that could be molded or shaped into a variety of items. Its biodegradability is derived from the fact that many bacteria that have the biosynthetic portion of the pathway also contain a biodegradative pathway (Anderson et at., Microbiological Reviews 54(4):450-472, 1990). Theoretically, thermoplastic items made from PHB could then be composted in landfills, where they can be degraded by both aerobic and anaerobic bacteria (Winton, Chemical Week, 55-57, Aug. 28, 1985). Commercialization efforts were initiated by W. R. Grace, but were halted when it became apparent that there were formidable technical difficulties to be overcome, and public interest in the project was low (Holmes, Phys. Technology 16:32, 1985).
PHB research languished until the 1970's, when the laboratories of H. G. Schlegel in Germany and E. A. Dawes in England undertook to elucidate the enzymological mechanism of PHB production. In a series of publications, both laboratories defined the pathway of PHB biosynthesis in Alcaligenes and Azotobacter, (Anderson et al., Microbiological Reviews 54(4):450-472, 1990; Jackson et al., Journal of General Microbiology 97:303-313, 1976; Oeding et al., Biochemical Journal 134:239-248, 1973; Ritchie, Biochemistry Journal 121:309-316, 1972; Schlegal et al., Antonie Van Leeuwenhoek 32:277, 1966; Senior et al, Biochemistry Journal 134:225-238, 1973; Ward et al., Journal of General Microbiology 102:61-68, 1977).
In the early 1980's interest in PHB was again stimulated when it was found that PHB is actually part of a family of polyesters, termed poly-.beta.-hydroxyalkanoates (PHAs) (Findlay et al., Applied and Environmental Microbiology 45(1):71-78, 1983). Loosely defined, PHAs are a family of polymerized fatty acid esters, in which the fatty acid monomer is normally from 4-10 carbons. PHAs that contain higher carbon-number fatty acids can be made into a more flexible thermoplastic, whereas PHAs containing lower carbon-number fatty acids tend to be more brittle (Byrom, Trends Biotechnology 5:246-250, 1987). For example, poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) is much more amenable to plastic film production than poly-3-hydroxybutyrate, which is a brittle plastic.
The environmental and commercial importance of PHAs lies in their potential to reduce the volume of solid waste. Although estimates vary widely, the best data available indicates that 7-10% of all landfill waste is plastic (Beardsley et al., Scientific American, 1988). This is the equivalent of millions of pounds of plastic disposed in this manner every day. Since the average life of such plastic can be as long as several hundred years, poly-.beta.-hydroxalkanoates offer distinct environmental advantages (e.g., a 0.07 mm-thick film of PHB degrades in 10 weeks in soil; Doi et al, Applied and Environmental Microbiology 55(11):2932-2938, 1989).
Unfortunately, PHA technology has not yet replaced petrochemical-based plastics because of the high cost of production. Currently, PHAs are being marketed for approximately $14 per pound, whereas petroleum-based plastics sell for less than $1 per pound (Winton, Chemical Week, 55-57, Aug. 28, 1985). The primary reason for the high cost of poly-.beta.-hydroxyalkanoates is the mode of production: fermentation times are as long as 100 hours, final PHA levels fluctuate, purification procedures are cumbersome and expensive, and substrate costs are inordinately high (Byrom et al., Trends Biotechnology 5:246-250, 1987). Therefore, before these plastics can find their way to the commodity marketplace, significant improvements are necessary.
The present invention overcomes previous difficulties of PHA production, and further provides other, related advantages.